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39 19 : 39 46 2005 Lactobacillus johnsonii 1 1 2 2 1 2 Effect of Fermented Milk Containing Lactobacillus johnsonii La1 on the Human Natural Immune System and Safety for the Excessive Administration Toshihiko YAMANO 1 *, Yoichi FUKUSHIMA 1, Mamiko TAKADA 2 and Hisakazu IINO 2 1* Nestlé Japan Ltd. Nutrition Business Group, 2 Showa Women s University 27 Lactobacillus johnsonii La1 L. johnsonii La1 1 10 9 cfu /120 g L. johnsonii La11 1 120 g 21 21 p 0.001 p 0.05 29 p 0.05 10 L. johnsonii La1 3 1 3 360 g 14 p 0.005 L. johnsonii La1 Abstract To elucidate the effect of fermented milk containing Lactobacillus johnsonii La1 on human blood phagocytic activity, a double-blind placebo controlled cross-over trial was conducted. Twenty-seven healthy Japanese volunteers were randomly divided into 2 groups, and either of test fermented milk containing L. johnsonii La1 (1 10 9 cfu /120 g) or placebo fermented milk without L. johnsonii La1 was administered daily for 21 days. At the end of the first period, blood phagocytic activity in the subjects administered test fermented milk increased significantly compared to that of before intake. Although phagocytic activity in the subjects administered placebo fermented milk also increased, the activity was low compared to that of the subjects administered test fermented milk. Blood phagocytic activities in the both groups were maintained at high levels after the 29 days wash-out period. At the end of the second period, phagocytic activity in the subjects administered test fermented milk was significantly higher than before intake. For confirmation of safety against excess dosage, 3 cups (360 g)/day of the test fermented milk was administered to 10 healthy persons for 14 days. Blood phagocytic activity significantly increased after administration of test fermented milk, and no adverse effects were observed in the medical check-up, blood and urine tests. These results suggest that ingestion of fermented milk containing L. johnsonii La1 is effective at reinforcing the natural immune system of humans without any safety concerns for excessive administration. Key words : lactobacilli ; healthy Japanese ; probiotics ; fermented milk ; blood phagocytic activity ; immunity ; safety 2004 4 9 140 0002 2 2 20 NYK Tennoz Building, 2 2 20 Higashi-shinagawa, Shinagawa-ku, Tokyo 140 0002, Japan

40 19 1 2005 16, 39, 40, 42 21, 24 Bifidobacterium Lactobacillus in vitro 14, 28, 31 2, 15 Lactobacillus johnsonii La1 Caco-2 HT29 4, 7 5, 41 34 13 L. johnsonii La1 9, 35 IgA 25, 27 L. johnsonii La1 1 1 10 9 cfu 9 L. johnsonii La1 L. johnsonii La1 3 Table 1. Composition of placebo and test fermented milk. Test Placebo per 120 g fermented milk Energy 102 kcal 102 kcal Protein 4.6 g 4.6 g Lipids 3.6 g 3.6 g Carbohydrates 12.9 g 12.9 g Na 60 mg 60 mg Ca 128 mg 128 mg S. thermophilus 1 10 7 cfu 1 10 7 cfu L. johnsonii La1 1 10 9 cfu 0 Final ph is below 4.5 1. 1 120 g L. johnsonii La1 1 10 9 cfu Streptococcus thermophilus LC1 L. johnsonii La1 Table 1 2. 27 21 59 8 19 44.2 10 24 55 5 5 41.1 3. a 27 A 13 5 8 42.2 B 14 3 11 46.2 2 Fig. 1 21 Day 1 I Day 1 Day 21 A B 1 1 21 Day 51 Day 71 A B 21 Day 0 22 50 72 4 b 10 1 3 14 Day 1 Day 0

Lactobacillus johnsonii La1 41 Fig. 1. Test schedule. A randomized double-blind placebo controlled cross-over trial was conducted. After 21 days of non-intake period, either test fermented milk containing L. johnsonii La1 (1 10 9 cfu /120 g) or placebo fermented milk was administered daily for 21 days in the test and placebo periods, respectively. 15 2 4. 8 100 µl FITC PHAGOTEST Orpegen Pharma 20 µl 1 10 9 /ml 20 1 37 0 10 DNA FACScalibur TM ; Becton Dickinson 0 5. MCH MCHC MCV A 1C SE-9000 AST ALT ALP LAP γ-gtp AMY HDL-C LDL-C CPK CRP Na Cl K Ca Fe 7600 ph US3100 6. SPSS 11.0 J Wilcoxon 5 Day 50 Day 0 p 0.001 Fig. 2 B Day 22 Day 0 p 0.001 A p 0.001 Day 22 B A p 0.05 Day 50 A B Day 0 p 0.001 p 0.05 A Day 72 Day 50

42 19 1 2005 Fig. 2. Effect of fermented milk containing L. johnsonii La1 on blood phagocytic activity. a : p<0.001 : Compared with Day 0. b : p<0.005 : Compared with Day 50. c : p<0.05 : Compared between groups A and B. MCV Table 2 Fig. 3. Effect of 3 times excessive administration of fermented milk containing Lactobacillus johnsonii La1 on blood phagocytic activity. a : p<0.005 : Compared with Day 0. p 0.005 B Fig. 2 Day 0 22 50 72 Fig. 3 Day 15 Day 0 p 0.005 Day 0 15 2 NK T B T B L. johnsonii La1

Lactobacillus johnsonii La1 43 Table 2. Analytical results of blood from subjects with excessive intake of fermented milk containing L. johnsonii La1. Test item standard value Day 0 Day 15 Blood components analysis Erythrocyte ( 10 4 /µl) 427 570 467.6 32.4 466.0 32.2 Hemoglobin 13.5 17.6 14.4 1.1 14.2 1.2 Hematocrit 39.8 51.8 42.1 2.4 42.5 2.5 Leukocyte ( 10 2 /µl) 39 98 47.8 8.3 52.0 17.9 Platelet ( 10 4 /µl) 13.1 36.2 23.8 3.5 23.7 2.6 MCH (pg) 28.0 34.6 30.7 1.2 30.4 1.2 MCHC ( ) 31.6 36.6 34.1 0.9 33.3 1.1 MCV (fl) 82.7 101.6 90.0 3.0 91.3 3.0 a Hemoglobin A 1C ( ) 4.3 5.8 4.8 0.3 4.9 0.3 Blood biochemical analysis Total protein (g/dl) 6.5 8.3 7.3 0.4 7.5 0.3 Albumin (g/dl) 3.8 5.3 4.4 0.2 4.4 0.2 Total bilirubin (mg/dl) 0.2 1.2 0.7 0.2 0.7 0.2 AST (IU/l) 8 38 20.2 4.3 20.5 3.5 ALT (IU/l) 4 43 18.6 10.5 20.1 9.2 ALP (IU/l) 110 354 184.0 61.5 189.8 61.5 LAP (IU/l) 30 70 51.0 6.7 52.4 7.4 γ -GTP (IU/l) 16 84 26.2 12.6 25.0 9.9 AMY (IU/l) 38 136 88.7 30.0 87.1 27.6 HDL-C (mg/dl) 40 77 68.3 18.7 65.7 18.8 LDL-C (mg/dl) 70 139 136.8 34.5 140.9 37.6 Insulin (µu/ml) 3.0 15.0 6.3 3.5 5.4 1.6 Total choresterol (mg/dl) 130 219 235.5 45.7 227.7 41.1 CPK (IU/l) 70 139 113.8 33.3 111.9 42.8 CRP (mg/dl) <0.6 0.1 0.0 0.1 0.0 Triglyceride (mg/dl) 30 149 235.5 45.7 227.7 41.1 Fasting blood glucose (mg/dl) 60 109 85.8 8.3 87.9 6.1 Uric acid (mg/dl) 3.5 7.7 5.8 0.8 5.5 0.7 Urea nitrogen (mg/dl) 8.0 22.0 11.6 1.8 11.6 2.0 Creatinine (mg/dl) 0.8 1.2 0.9 0.1 0.9 0.1 Na (meq/l) 135 180 141.7 1.9 142.5 0.8 Cl (meq/l) 98 110 102.0 1.3 102.8 1.4 K (meq/l) 3.5 5.3 4.1 0.2 4.1 0.4 Ca (meq/l) 8.4 10.2 9.5 0.3 9.1 0.3 Fe (meq/l) 58 188 126.4 26.5 114.3 29.8 a : p<0.01 Compared with Day 0. 1 1 21 29 Day 50 Day 0 29 S. thermophilus 9

44 19 1 2005 Day 22 L. johnsonii La1 GALT M 10, 36 L. johnsonii La1 TGF-β mrna Caco-2 in vitro 18 IFN-γ IL-12 NK 19, 20 LTA 1, 6, 23 L. johnsonii La1 LTA HT29 44 L. johnsonii La1 LTA Caco-2 3, 11, 30 M L. johnsonii 22 L. acidophilus 32, 33 L. johnsonii La1 LTA L. johnsonii La1 IFN-γ IL-12 S. thermophilus S. hermophilus LTA LTA 17, 20 LTA 29 LTA L. johnsonii La1 8, 37 Bifidobacterium Lactobacillus 12, 26, 43 Bifidobacterium Lactobacillus Lactobacillus L. johnsonii La1 L. johnsonii La1 1 1 10 9 cfu 9 L. johnsonii La1 1 3 14 1 1 10 9 cfu L. johnsonii La1 1 1 10 9 cfu 80

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